Method for preparing engineered tissue

ABSTRACT

A method for preparing a human or animal tissue by applying a compressive force to a stack of sheets of living tissue thereby inducing adjacent layers to fuse or adhere to each other. The force is applied in direction normal to the surface of the tissue. A multi-layer tissue produced by the method described above can also possess at least two different types of sheets and/or consist essentially of between two and twelve sheets of living tissue. The method can also be used to prepare a planar tissue that can further be incorporated in a multi-layer tissue construct. The methods and tissues described herein are useful for the preparation of engineered tissues.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the 371 National Phase of International ApplicationNo. PCT/CA2003/001079, filed Jul. 16, 2003, which was published inEnglish under PCT Article 21(2) as International Publication No. WO2004/007699. This application further claims the benefit under 35 U.S.C.§ 119 (e) of U.S. provisional application No. 60/396,004 filed Jul. 16,2002. All of these applications are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to engineered tissue. In particular, thepresent invention relates to methods for making engineered tissue fromsheets of living tissue.

BACKGROUND OF THE INVENTION

Tissue engineering may be used to recreate tissues and organs forgrafting onto patients. Engineered tissues and organs can also serve asin vitro models. A variety of tissue engineering techniques are known,including tissue in-growth, seeding of cells on artificial orbiodegradable scaffolds, and collagen gels. Among them, a new method oftissue engineering, known as the “self-assembly” method, has emerged(L'Heureux et al.; Michel et al.; Pouliot et al.). In the self-assemblymethod, cells are induced to secrete and organise an extracellularmatrix and thereby form a sheet of living tissue. The self-assemblymethod takes advantage of the fact that fibroblasts can produce asuitable extracellular matrix when grown in the presence of ascorbicacid. To create multi-layer tissue constructs, sheets of living tissuecan be stacked upon each other, folded upon themselves, or rolled on atubular support.

The development of tissue engineering methods to produce reconstructedtissues has focused on the optimization of morphological andhistological properties of the reconstructed tissues. Most tissueengineering research has focused on optimizing techniques for growingsheets of tissue. However, in many cases, it would be desirable to makereconstructed tissue comprised of several layers of tissue and/or layersof more than one type of tissue. Multi-layer tissue constructs arethicker and therefore stronger and since multi-layer tissue constructscan comprise more than one sheet of living tissue, they can be designedto more closely resemble the tissues that they are intended to replace.However, in order to create useful multi-layer tissue constructs, it isessential to be able to fuse adjacent layers of cell tissue together sothat the layers are bonded together as firmly and reliably as possibleand resist separation. If these layers of tissue are not fused togetherwell, they may separate or come apart over time, for example, duringhandling or in the body of a patient.

Thus, it would be desirable to have a method for preparing multi-layeredengineered tissue constructs with improved fusion between adjacentlayers of tissue.

DESCRIPTION OF BACKGROUND ART

Ye et al. teach that mechanical stress can enhance the synthesis andsecretion of the principal extracellular matrix protein, collagen, byfibroblasts, thereby increasing the mechanical strength of the stiffnessof reconstructed tissue. Ye et al. describe a method for applyingmechanical stress wherein sheets of fibroblast cells are mounted onframes to apply tension. However, the reconstructed tissues produced bythis method have significantly less collagen than does native tissue andtherefore would not be expected to have mechanical properties thatresemble native tissue.

Kanda et al. teach that mechanical stress induces cell orientation andphenotypic modulation of cultured smooth muscle cells.

L'Heureux et al. describe a method of making a tissue-engineered bloodvessel (TEBV) by wrapping sheets of living tissue around a tubularsupport.

Lopez-Valle et al. describe the use of a continuous (as opposed topunctuated or discontinuous) anchor made of porous glass microfibermaterial, the pores of which trap collagen fibers and thereby induceorganization of extracellular matrix and orientation of cells.

SUMMARY OF THE INVENTION

The current invention provides a method for improving the fusion betweenadjacent layers of living tissue in a multi-layered engineered tissueconstruct. The current invention further provides a method for making asheet of living tissue suitable for use in preparing a multi-layeredengineered tissue construct. Multi-layered reconstructed tissuesproduced by this method have improved fusion between layers of tissueand the layers of tissue are less likely to separate during subsequentmanipulation.

Thus, in one aspect, the invention provides a method for preparing ahuman or animal tissue from at least one sheet of living tissue, themethod comprising the steps of: (a) arranging the at least one sheet ofliving tissue to form a multi-layer stack of living tissue; and (b)applying a compressive force in a direction normal to the surface of themulti-layer stack of living tissue with a force-applying means at apressure and for an amount of time sufficient to compress layers oftissue together for inducing adjacent layers of tissue to fuse or adhereto each other.

A particular preferred embodiment provides a method for producing atissue by forming a multi-layer stack of living tissue arranged on asubstantially flat support. More preferably, the multi-layer stack isformed by superimposing two or more sheets of living tissue and/or byfolding a sheet of living tissue upon itself.

In a further embodiment, the method comprises another step where themulti-layer stack is then anchored to a substantially flat supportsurface with moveable anchors comprising weights or ingots, wherein theanchors are of a suitable weight for (1) applying sufficient tensionacross the sheet of living tissue to prevent shrinkage and/or maintaincellular differentiation and/or induce orientation of cells in at leastone sheet of living tissue and (2) allowing contraction of at least onesheet of living tissue once a predetermined threshold of tension isexceeded across the sheet of living tissue. A force is then appliednormal to the surface of the layers of tissue by way of a weighteddevice suitable for applying evenly distributed pressure to the surfaceof the multi-layer stack of tissue, the weighted device being at leastpartially permeable to culture medium, for inducing adjacent layers oftissue to fuse to each other.

In a preferred embodiment, the force-applying means in step (b) of themethod comprises a weight device suitable for applying substantiallyevenly-distributed pressure to said multi-layer stack of living tissue,the weight device being at least partially permeable to tissue-culturemedium.

In a further embodiment, the multi-layer stack of step (a) of the methodis formed by rolling a sheet of living tissue on a tubular support and,more preferably, the force-applying means of step (b) comprises atissue-culture medium permeable elastic sleeve.

There are many different types of sheets of living tissues that can beused with the method described herein. In an embodiment, the methodutilizes a biopsy as a sheet of living tissue. In another embodiment,the method utilizes cells cultured in vitro as a sheet of living tissue.More preferably, the cells are embryonic stem cells, post-natal stemcells, adult stem cells, mesenchymal cells, hepatocytes, Islet cells,parenchymal cells, osteoblasts and other cells forming bone orcartilage, and nerve cells. The mesenchymal cells can either befibroblasts, interstitial cells, endothelial cells, smooth muscle cells,skeletal muscle cells, myocytes, chrondocytes, adipocytes,fibromyoblasts, or ectodermal cells. In a further embodiment, the sheetof living tissue can also be a skin tissue, a corneal tissue, a cardiacvalve tissue, a connective tissue and/or a mesenchymal tissue.

In another aspect, the invention also provides a multi-layer tissue madeaccording to the method described herein, wherein the multi-layer tissuecomprises at least two different types of sheets of living tissue. In apreferred embodiment, the tissue consists essentially of between two andtwelve sheets, more preferably of between three and nine sheets. Inanother embodiment, the tissue has a thickness of between about 0.01 mmto about 0.5 mm, more preferably of between about 0.03 mm to about 0.45mm.

In a further aspect, the invention provides a method for preparing aplanar human or animal tissue suitable for use in making a multi-layertissue construct from at least one sheet of living tissue, the methodcomprising the steps of: (a) arranging said at least one sheet of livingtissue on a substantially flat support surface; and (b) anchoring saidat least one sheet of living tissue to the support surface with anadjustable anchor-means comprised of a multiplicity of spaced apartanchors, wherein the anchors are suitable for (1) applying sufficienttension across the sheet of living tissue to prevent shrinkage and/ormaintain cellular differentiation and/or induce orientation of cells insaid at least one sheet of living tissue and (2) allowing contraction ofsaid at least one sheet of living tissue once a predetermined thresholdof tension is exceeded across the sheet of living tissue.

Preferably, the adjustable anchor-means is comprised of discretemoveable anchors such as weights or ingots. Alternatively, theadjustable anchor means may also comprise a moveable frame or amultiplicity of moveable anchors mounted on a frame.

In a particular preferred embodiment, a planar construct can be made byforming a multi-layer stack of living tissue by superimposing two ormore sheets of living tissue or by folding a sheet of living tissue uponitself. The multi-layer stack is then anchored to a substantially flatsupport surface with moveable anchors comprising weights or ingots,wherein the anchors are of a suitable weight for (1) applying sufficienttension across the sheet of living tissue to prevent shrinkage and/ormaintain cellular differentiation and/or induce orientation of cells inat least one sheet of living tissue and (2) allowing contraction of atleast one sheet of living tissue once a predetermined threshold oftension is exceeded across the sheet of living tissue. A force is thenapplied normal to the surface of the layers of tissue by way of aweighted device suitable for applying evenly distributed pressure to thesurface of the multi-layer stack of tissue, the weighted device being atleast partially permeable to culture medium, for inducing adjacentlayers of tissue to fuse to each other.

In a further embodiment, the sheet of living tissue used to prepare theplanar construct is obtained by culturing cells in vitro.

In yet another aspect, the invention provides a planar multi-layertissue consisting essentially of between two to twelve sheets of livingtissue obtained by the method described herein.

In another particular embodiment, a tubular construct can be made byforming a multi-layer stack of living tissue by rolling a sheet ofliving tissue onto itself, for example with the aid of a tubularsupport. A culture medium permeable elastic sleeve can be used tocompress the layers of tissue in the multi-layer stack of living tissuetogether for inducing the adjacent layers of tissue to fuse to eachother.

Other embodiments and advantages of the invention will become apparentfrom the detailed description to follow, together with the accompanyingdrawings.

All publications, figures, patents and patent applications cited hereinare hereby expressly incorporated by reference in their entirety for allpurposes to the same extent as if each was so individually denoted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a preferred method of making a planar tissue. (A) Afirst sheet of living tissue (1) is arranged on a substantially flatsupport surface and anchored peripherally with weights or ingots (2); asecond sheet of tissue (3) is superimposed on the first sheet of tissue,and the weights or ingots (2) are moved, one-by-one, and placed on thesecond sheet, thereby anchoring the superimposed sheets. (B) A sponge(4) that has been cut to fit within the ingots is placed on top of themulti-layer tissue construct (5) so formed; and spaced-apart weights (6)are placed on the sponge.

FIG. 2 illustrates a preferred method of compressing a tubular tissueconstruct. An elastic sleeve (7) is placed around a hollow tube (8)using its tapered end (9). The hollow tube (8) is larger than a tissueconstruct (10) which has been rolled around a mandrel (11). The hollowtube (8) is then placed around the tissue construct (10). The elasticsleeve (7) is transferred from the hollow tube (8) to the tissueconstruct (10) by gently displacing the tube (8) in one direction andthe tissue construct (10) in the opposite direction.

FIG. 3 is a microscopic view of the tissue made according to the methodof the invention, after maturation. The tissue is assembled from ninesheets of living tissue containing fibroblasts and extracellular matrixconstituents. Magnification 20×, scale bar 50 μm.

FIG. 4 graphs results of cyclic stress-strain test on a three-layertissue construct made as described in Example 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, it has been found thatapplication of a compressive force normal to the plane of a sheet oftissue enhances fusion between adjacent layers of sheets of tissue.Compression improves contact between layers of tissue and encouragesfusion of the layers of tissue. By way of example, a weighted device(for example, a sponge upon which spaced apart weights are placed) maybe applied to a stack of two or more superimposed planar sheets oftissue, thereby applying a force normal to the plane of the sheets oftissue (see FIG. 1B). In another example, a suitably sized elasticsleeve can be fitted over a multi-layer stack of tissue made by rollingat least one sheet of tissue onto a tubular support, whereby the elasticsleeve applies pressure normal to the two-dimensional plane of the sheetof tissue (see FIG. 2).

Preferably, the compressive force or pressure is applied evenly on theentire tissue surface. Therefore, it is preferable that a device adaptedto the shape of the tissue be used to induce the fusion. The amount ofpressure applied to the surface of the tissue stack can be adjustedaccording to the needs of the engineered tissue. This pressure isapplied for a period of time sufficient to allow the complete fusion ofthe tissue layers, preferably between 24 hours to 7 days.

It is also preferable that the device used to induce pressure to thesurface of the tissue be permeable to culture media in order to allowthe nutrition of the living cells. An acceptable way to generate thispressure on a flat tissue is to lay a semi-rigid sponge on the top ofthe tissue stack. Additional weight (for example, one or more solidingots) can be distributed on top of the sponge to obtain the desiredamount of pressure on the tissue (see FIG. 1B). Of course, any othersystem using mechanical or hydraulic pressure could be used to providethis compression.

In the example of a tubular or cylindrical construct, the compressingdevice should preferably apply equal pressure on the external surface ofthe construct. In this particular case, a good way to compress thetissue is to apply an elastic and permeable sleeve around the construct(see FIG. 2). The size and the elasticity of the sleeve can be adjustedto give the appropriate pressure for a given tissue.

To wrap the sleeve around the tissue and remove it without damage, aninstallation device may be used (see FIG. 2). The elastic sleeve (7) ismounted on a rigid hollow tube (8) using the tapered end of the tube(9). The tube, having an internal diameter slightly larger than theconstruct, is then passed over the tubular tissue (10) rolled around amandrel (11). The end of the sleeve is then anchored to the mandrel andthe hollow tube is carefully removed by pulling from the opposite side,gently depositing the sleeve on the tissue. To remove the sleeve withoutdamaging the tissue, it may be for example carefully cut or unsewn.

It is known that mechanical stress may be used to induce cellularorientation and phenotypic modulation of cultured smooth muscle cells(Kanda et al.; Germain et al.). Thus, appropriate forces may be appliedto maturating tissue in order to induce fiber orientation. Such forcesmay also prevent shrinkage and maintain the desired celldifferentiation.

As an example, it has been shown that a continuous anchor, such as aframe or a ring of glass microfiber that circumscribes or encircles atissue, may be used to induce cellular orientation (Ye et al.; Kanda etal.). The induction of cell orientation is thought to occur because thecontinuous anchor mechanically restricts the spontaneous contraction ofthe maturing cultured tissue, thereby creating a mechanical stress ortension across the tissue that induces cell orientation.

The underlying mechanism of the orientation response has not been wellelucidated (Kanda et al.). However, when a continuous anchor is used,the tension across the tissue continues to build as the tissue maturesand can reach levels that are detrimental to the health of the cells inthe tissue, reducing viability of cells contained in the tissue andthereby producing an inferior tissue construct. Therefore, a continuousanchor, such as a rigid frame, may not be suitable for use with sometissue types, i.e. those tissues that can create a lot of tension asthey mature.

The current invention provides an improved method of anchoring maturingcultured tissues, the method comprising an adjustable anchor means,preferably comprising a multiplicity of spaced apart anchors (such asmoveable weights or ingots), wherein the anchors are suitable for (1)applying sufficient tension across the sheet of living tissue to preventshrinkage and/or maintain cellular differentiation and/or induceorientation of cells in at least one sheet of living tissue and (2)allowing contraction of at least one sheet of living tissue once apredetermined threshold of tension is exceeded across the sheet ofliving tissue (for an illustration see FIG. 1A).

The anchor means is “adjustable” in that once the tissue has built up atension higher than the maximum tension that can be held by the anchors(i.e. weights or ingots), the tissue can spontaneously contract and theanchors will be pulled along with the contracting tissue. Thus, thetension across the tissue cannot continue to build up when an adjustableanchor means as described is employed. The maximum tension that canbuild up across the tissue can be controlled by choosing suitableanchors (for example weights or ingots of a certain weight and number,or an adjustable frame that is designed to move in response to a certaintension or force). Thus, it is possible to optimize the amount oftension for any given tissue, for example, to enhance viability of cellsin the tissue.

Anchors according to the current invention may be “discontinuous” or“punctual”. A “discontinuous” or “punctual anchor” is a device thatanchors a tissue substantially at a point in space. In contrast, in thecontext of the present invention, the term “continuous anchor” refers toa device for securing a tissue around its entire perimeter (such asdescribed by Lopez-Valle et al.).

The anchors of the present invention may be “moveable” in that they caneasily be placed on a sheet of tissue or removed therefrom.

For making planar sheets of living tissue for use in making multi-layertissue constructs, it is preferred that anchors are arranged to form aclosed perimeter near the edge of a sheet of tissue. This geometryinduces cells and extracellular matrix fibers in the sheet of tissue toorient in the two dimensions of the plane of the sheet of tissue. Thisorientation of cells and extracellular matrix may be beneficial forfusion of adjacent layers of sheets of tissue and may also improvecertain functional properties of the tissue. For example, in FIG. 1A, afirst sheet of living tissue (1) is disposed on a flat surface andingots (2) keep the first sheet in place. A second sheet of livingtissue (3) is placed on top of the first sheet. Ingots are displacedfrom the first sheet and are arranged on top of the second sheet toanchor the stack of sheets. The ingots are arranged to follow theperimeter of the stack of living sheets. The ingots also provide adiscontinuous mechanical force to the living sheets allowing cellulardifferentiation and contraction. The process may be repeated to obtain amulti-layer tissue construct.

The current invention provides the use of a multiplicity of spaced apartweights or ingots as anchors for applying mechanical force to tissue ina punctuated or discontinuous manner along the edge of the sheet ofliving tissue. If the weights or ingots are arranged very close to eachother or so as to contact each other, they may displace each othersomewhat when the tissue contracts. The amount and direction ofmechanical force applied to a sheet of tissue can be controlled byvarying the number, weight and position of the weights or ingots. Hence,it is possible to optimize or fine-tune the mechanical force conditionsfor any particular size or type of tissue.

The current invention is in contrast to the continuous anchor made ofporous glass microfiber material described by Lopez-Valle et al. Aporous continuous anchor like that described in Lopez-Valle et al. isnot easily moved, removed or adjusted, and as a result, does not provideone with the ability to fine-tune the application of mechanical force toa tissue.

Weights or ingots for use as anchors according to the current inventionmay be made from any material that does not interfere with thedevelopment or differentiation of cells in the sheet of living tissue,such as stainless steel. Magnets or metal ingots coated with Teflon™ orany polymer material known in the art to be compatible with tissueculture may also be used. Suitable weight values for the weights oringots for use with a tissue type can be determined empirically.Preferably, weights are chosen so that cell orientation and/ordifferentiation are induced.

The foregoing technique of using adjustable/moveable anchors andcompression to fuse tissues together also may be used for producingthree-dimensional tissue constructs.

Preparation of Sheets of Living Tissue

Sheets of living tissue for use in making multi-layered reconstructedtissue in accordance with the current invention may be obtained frombiopsy or may be made using any known techniques. In the case wheresheets of living tissue of mesenchymal origin are prepared using tissueengineering techniques, a preferred method is the self-assemblyapproach, which allows normal cell-cell and cell-extracellular matrixinteractions. In addition, the self-assembly approach allows thesecretion of important natural growth factors and cytokines, and theformation of a mature connective tissue necessary for functionality ofthe tissue and for the cells in the tissue to remain metabolicallyactive and undergo normal mitosis.

The subsections below describe preparation and use of human engineeredtissue in vitro. However, the invention is not limited to humanengineered tissue and extends to animal tissue and engineered tissuewith transformed (human and non-human) cells as well.

Cell Source

A variety of cells can be used in the human engineered tissue of thepresent invention. Preferred cell types include embryonic stem cells,amniotic fluid cells, post-natal stem cells, adult stem cells,mesenchymal cells, especially fibroblasts, interstitial cells,endothelial cells, smooth or skeletal muscle cells, myocytes (musclestem cells), chrondocytes, adipocytes, fibromyoblasts, and ectodermalcells, including ductile and skill cells, hepatocytes, Islet cells,cells present in the intestine and other parenchymal cells, osteoblastsand other cells forming bone or cartilage, bone marrow cells and bloodcells. In some cases it may also be desirable to include nerve cells.

Cells can also be genetically engineered to provide additional or normalfunction. Methods for genetically engineering cells with retroviralvectors, polyethylene glycol, and other methods known to those skilledin the art can be used.

Cells may be autologous, allogeneic or xenogeneic, however autologous orallogeneic cells are preferred. Immunologically inert cells, such asembryonic or fetal cells, stem cells, and cells genetically engineeredto avoid the need for immunosuppression may also be used. Methods anddrugs for immunosuppression are known to those skilled in the art oftransplantation.

In some embodiments, cells are obtained by biopsy and dissociated usingstandard techniques, such as digestion with a collagenase, trypsin orother protease solution. For example, the dermal layer of a skin biopsycan be digested with collagenase according to the method of Germain andAuger. After the digestion of the dermal fragments, mesenchymal cellsare harvested following centrifugation and expanded in cell culturemedia. All cell cultures are used between their fourth and eightpassages, and kept incubated at 37° C. and 8% CO₂. Cells can be easilyobtained through a biopsy anywhere in the body, for example, skeletalmuscle biopsies can be obtained easily from the arm, forearm, or lowerextremities, and smooth muscle can be obtained from the area adjacentthe subcutaneous tissue throughout the body. The biopsy can be readilyobtained with the use of a biopsy needle, a rapid action needle whichmakes the procedure extremely simple and almost painless. Cells may alsobe procured from, for example, blood vessels, blood, such as umbilicalcord blood, valves and discarded tissues, such as foreskins and tissueobtained during esthetic or cosmetic surgical procedures.

Fibroblasts, such as dermal fibroblasts or adventitial fibroblasts, maybe used. Fibroblasts are easily available, and they are the primarycollagen secreting cells in connective tissues. Dermal fibroblasts aretypically harvested from normal adult skin specimens removed duringreductive breast surgery, or from neonatal foreskin. The potential ofhuman fibroblasts for cardiovascular application is enormous for bothallogeneic and autologous grafts since cells contained in onesquare-inch of foreskin can be used to grow many acres of tissue.

Preparation of a Sheet of Living Tissue

The engineered tissue of the present invention is formed from at leastone sheet of living tissue. Each sheet of living tissue is comprised ofcells and an endogenous extracellular matrix. The extracellular matrixis secreted by cells, such as mesenchymal cells, embryonic stem cells oradult stem cells, to name a few. When mesenchymal cells, such as dermalfibroblasts, are cultured in a planar culture substratum usingL-ascorbic acid or a phosphate derivative of L-ascorbic acid (e.g. Asc2-P), serum, and growth factors, they show an abundant synthesis ofextracellular matrix proteins. This creates the basis of the endogenousextracellular matrix. L-ascorbic acid plays an important role since itis a cofactor for the hydroxylation of proline and lysine residues incollagen (Hata and Senoo), and also it increases both the rate oftranscription of procollagen genes and stability of procollagen mRNA(Tajima and Pinnell). The extracellular material is comprised ofdifferent proteins, such as collagen type I, other collagen types(fibrillar and non-fibrillar), elastin, fibrillin, glycosaminoglycans(such as decorin), growth factors, and glycoproteins, to name a few.

In the context of the present invention, the resulting living tissueformed from the cells and the extracellular matrix as described above iscalled a “sheet of living tissue”.

An exemplary embodiment of methodology for generating such sheets ofliving tissue is described in U.S. Pat. No. 5,618,718 by Auger et al. Insummary, Auger et al. describe that dermal fibroblasts, at aconcentration equivalent to 10⁴ cells/cm², are plated into 75 cm²sterile Petri dishes. Cell medium is supplemented with a 3:1 DMEM andHam's F12 modified medium, fetal bovine serum, penicillin andgentamicin, and with an ascorbic acid solution. For example, a finalascorbic acid solution between 50-100 μg/ml can be used every other day.Culture conditions are kept at 92% air and 8% CO₂ at full humidity.Culture time is approximately three weeks. At the end of the maturationtime, the sheet of living tissue spontaneously detaches from thesubstratum.

It can be appreciated that a variety of methods can be used to preparethe sheets of living tissue (e.g. Auger et al.; Ye et al.; L'Heureux etal.; Michel et al.; Pouliot et al.) and the present invention is notlimited in scope by using one particular shape (i.e. thickness andsize), cell type, origin, age, maturation time, component concentration,and culture conditions to generate the sheet of living tissue.

Preparation of Engineered Tissue

The engineered tissue of the present invention is formed fromsuperimposing a plurality of individual sheets of living tissue. In anembodiment, the number of sheets varies between two and twelve. Asdescribed above, the sheets of living tissue are comprised of anextracellular matrix secreted by cells, such as mesenchymal cells. Theextracellular matrix is produced with many in vivo-like propertiesincluding supramolecular organization of collagen. Collagen is not onlyprocessed, but is also cross-linked efficiently and the collagen fibrilsare assembled into bundles. When the sheet is layered upon itself, forexample by folding or wrapping, or a plurality of sheets are stacked orsuperimposed, a three-dimensional construct having desired structuralcharacteristics is formed in culture.

In some embodiments, the sheets of living tissue are stacked in a cellculture dish, either directly superimposed or in an overlapping fashion.By overlapping, tissues of various shapes may be formed. For example,rectangular sheets of living tissue may be arranged in an overlappingfashion to create a circular layered tissue construct. Or, irregularlyshaped cell cultured sheets may be stacked in a manner to form aregularly shaped tissue. In addition, the individual sheets may bestacked in the same orientation or the orientation of the sheets may bevaried to create specific effects in the resulting tissue.

Alternatively or in addition, one or more sheets of living tissue can befolded to form a multitude of layers. For example, one sheet may befolded upon itself in an accordion-type fashion or in repeated halves tosuperimpose portions of the sheet upon itself. Or, two or more sheetsmay be stacked to form a multi-layer stack of tissue, which multi-layerstack of tissue may then be folded upon itself to create even morevariety of layering.

Alternatively or in addition, a wrapping technique, such as wrapping asheet around itself in the style of a cinnamon roll, can be used tocreate a multi-layer stack of tissue. It is possible to combinedifferent/many techniques, for example by first creating a multi-layerstack of tissue by (1) layering more than one sheet of tissue and/or (2)folding a sheet or a multi-layer stack of sheets of tissue on itself,then wrapping the multi-layer stack of (1) or (2) around itself.

When layering, the sheets of living tissue are held together by surfaceadhesion between the sheets. Any number of sheets of living tissue maybe used, preferably five or more, more preferably seven or more, andmore preferably, nine, ten or eleven or more. The sheets are delicatelyhandled with forceps and superimposed or otherwise assembled to form thehuman engineered tissue construct. By maintaining this construct inculture medium supplemented with ascorbic acid under conditions similarto those described in Huynh et al., the sheets of living tissue willfuse together to form a human engineered tissue resembling thecorresponding mature tissue (FIG. 3).

For some applications, it is preferred that the resulting reconstructedtissue comprises more than one type of sheet of living tissue. Forexample, a reconstructed tissue suitable for use as a skin graft maycomprise sheets of a dermal equivalent and epidermal equivalent. Areconstructed tissue suitable for use as a corneal graft shouldcomprises the following layers: an epithelial equivalent; a stromalequivalent; and endothelial equivalent.

Maturation time of the construct will depend on the nature of the tissueand the specific mechanical properties desired. For example, it has beenfound that mechanical strength of certain tissue constructs plateauafter seven weeks of maturation (L'Heureux et al.). For any given tissueconstruct, the maturation time necessary to obtain optimal functionalitymay be readily determined using routine methods known in the art.

Generally, the engineered tissue is thin enough to allow oxygen deliverythrough its surfaces to maintain metabolic needs yet thick enough toprovide desired functionality. The current embodiments of the engineeredtissue of the present invention are avascular, wherein the tissue doesnot include a microvasculature to deliver oxygenated blood to thetissue. Therefore, the tissue relies on oxygen diffusion from itssurfaces to sustain the tissue. Due to oxygen diffusion limitations, thetissue thickness is currently an important consideration. (Weind etal.). The thickness of the engineered tissue may be controlled bychoosing the number of sheets of living tissue used. The engineeredtissue may have a thickness ranging from approximately 0.01 mm to 0.5mm; more preferably between 0.03 mm to 0.45 mm. Preferred thickness willvary depending on the tissue type, intended function of the engineeredtissue and the type of cells used.

If required, mature tissue constructs may be cut into a desired shapeusing any suitable method, such as die cutting and template cutting.

In an embodiment, cells from many different species and/or transformedcells can be used. Since it is contemplated that many applications ofengineered tissue will concern treatment of human patients, humanengineered tissue is especially preferred.

Preconditioning

If desired, the tissue-engineered construct may be preconditioned toreduce shrinkage, for example as described in the US application byLafrance et al.

Although various embodiments of the invention are disclosed herein, manyadaptations and modifications may be made within the scope of theinvention in accordance with the common general knowledge of thoseskilled in this art. Such modifications include the substitution ofknown equivalents for any aspect of the invention in order to achievethe same result in substantially the same way. Numeric ranges areinclusive of the numbers defining the range. In the claims, the word“comprising” is used as an open-ended term, substantially equivalent tothe phrase “including, but not limited to”. The following examples areillustrative of various aspects of the invention, and do not limit thebroad aspects of the invention as disclosed herein.

EXAMPLE 1

Preparation of a Reconstructed Multi-Layered Human Tissue Construct fromSheets of Living Tissue Containing Fibroblasts and Extracellular MatrixConstituents.

The following example describes a method for preparing a reconstructedmulti-layered human tissue construct from sheets of living tissuecontaining fibroblasts and extracellular matrix constituents accordingto the present invention (see FIG. 1 for illustration of the method).All of the procedures described below are done under sterile conditions,preferably using a sterile flow hood. It can be appreciated that avariety of methods can be used to prepare the multi-layered tissueconstruct and this example is not intended to limit the scope of thisinvention to the number of sheets of tissue superimposed, to oneparticular shape (i.e., thickness and size), cell type, origin, age,maturation time, component concentration, and culture conditions togenerate the multi-layered human tissue construct. One skilled in theart can readily appreciate that various modifications can be made to themethod without departing from the scope and spirit of the invention.

In this example, to produce a sheet of living tissue, 750,000 viablesub-cultured human skin fibroblasts are seeded in a standard 75 cm²sterile petri dish for a final seeding density of 10⁴ cells/cm². Cellsare fed with culture medium (DME containing 10% fetal calf serum (FCS),100 IU/ml penicillin and 25 μg/ml gentamicin), and cultivated for 4weeks to form sheets that can be manipulated. The culture medium ischanged three times per week. A freshly prepared solution of ascorbicacid is added each time the medium is changed to obtain a finalconcentration of 50 μg/ml of ascorbic acid. During culture, cells arekept in a humidified atmosphere (92% air and 8% CO₂).

After the sheets of tissue are formed, they are peeled from the dishes,and three separate sheets of living tissue are superimposed using thefollowing technique. A first sheet of living tissue is put into a petridish and culture media is added over the sheet to keep it wet and tohelp to spread it. Stainless steel ingots (approximately 1 mm×2 mm×8 mm)are placed around the tissue sheet perimeter to keep the tissue sheetanchored and stretched to its maximal area on the surface of the petridish. Another sheet of tissue is then placed on top of the first sheetof tissue. One by one, the ingots are carefully pushed aside from thefirst sheet and other ingots were placed around the tissue sheetperimeter of the second layer, spreading it over the first sheet oftissue. These steps were repeated to obtain a three-layered tissueconstruct.

A semi-rigid sponge permeable to the culture media is then cut to fitthe size of the tissue construct between the ingots and applied to thesurface of the construct (see FIG. 1B). The sponge should closely fitthe perimeter delimited by the ingots, but not overlap or exceed it.Ingots are then evenly distributed on the sponge surface to put someweight on it (in this case, 11 g/40 cm² [0.275 g/cm²]). The sponge aswell as the ingots are removed 24 hours to 7 days following thestacking.

Seven days after the stacking of the sheets of tissue, threethree-layered tissue constructs were superimposed to form the finalnine-layered tissue construct using the same technique as describedabove. The constructs were further incubated for up to 8 weeks andculture medium refreshed 3 times a week. The tissue constructs are thenready for shipment processing.

EXAMPLE 2

Microscopic Analysis of the Tissue Construct

The tissue construct is prepared according to the procedure described inExample 1. In this example, the tissue construct is assembled from ninesheets of living tissue.

Biopsies of the living tissue construct are first fixed in Bouin's™solution. Cross-sections of the fixed tissue are embedded in paraffin.The cross-sections are stained with Masson's trichrome. Microscopicobservations are done on a Nikon TS100™ microscope at 20× magnification.

FIG. 3 shows a microscopic cross-section of the tissue constructobtained after the stacking and maturation of 9 sheets of living tissuecontaining fibroblasts and extracellular matrix constituents. This lightmicroscopy demonstrates a tissue construct resembling that of a nativetissue with dense extracellular matrix. In addition, the 9 superimposedsheets of living tissue have fused together to form one single tissueconstruct.

EXAMPLE 3

Biomechanical Properties of the Tissue Construct

Mechanical properties of the tissue are determined by simple tensiletests and cyclic tensile tests. These tests are performed using aTytron™ 250 MicroForce Testing System, (MTS Systems Corporation). Thismachine allows the loading and unloading of the tissue at differentspeed rates, and makes data acquisition of the stress and thedeformation applied to the tissue. Both tests are made on 7.9 mm widthtissue slices, for a total of three slices per tissue. Traction speed isset to 1 mm/s for both tensile and cyclic tests.

A simple tensile test consists in stretching the tissue until the loadbecomes high enough to break it. It allows the measure of the modulus ofelasticity and the ultimate tensile strength of the tissue. These twovalues give the relative stiffness and resistance of the tissue.

Cyclic tensile tests allow determination of the percentage of plasticdeformation of the tissue following a stretch. The percentage of plasticdeformation evaluates the capacity of a tissue to recover its originalshape after a load is applied to it. The cyclic tensile test isperformed by stretching the tissue until 10% of the ultimate tensilestrength of the tissue is reached. Then the traction is stopped and theload removed from the tissue at the same rate it was applied previously.This result gives the amount of irreversible deformation the tissue hadto endure while it was stretched.

FIG. 4 graphs cyclic stress-strain test on a mature three-layer tissueconstructs made as described in Example 1. The tissue construct isresistant to tensile stress. It also has the capacity to recover itsoriginal shape after a 10% strain.

Throughout this application, various references are referred to describemore fully the state of the art to which this invention pertains. Thedisclosures of these references are hereby incorporated by reference intheir entirety into the present disclosure for all purposes.

REFERENCES

-   Auger et al. U.S. Pat. No. 5,618,718 issued Apr. 8, 1997.-   Germain and Auger. “Tissue engineered biomaterials: biological and    mechanical characteristics”, In: Wise, Trantolo, et al. editors:    “Encyclopedic handbook of biomaterials and bioengineering”, NY,    N.Y.: Marcel Dekker Inc., 1995, pp. 699-734.-   Germain et al. Patent application WO 03/045458, published Jun. 5,    2003.-   Huynh et al. U.S. Pat. No. 5,928,281 issued Jul. 27, 1999.-   Hata and Senoo. J Cell Physiol. (1989) 138,8-16.-   Kanda et al. ASAI0 Journal (1993) 39, M686-90.-   Lafrance et al. US application Serial No. 20030027332 published Feb.    6, 2003.-   L'Heureux et al. The FASEB Journal (1998) 12, 47-56.-   Lopez-Valle et al. British Journal of Dermatology (1992) 127,    365-371.-   Michel et al. In Vitro Cell Dev Biol Anim (1999) 35, 318-26.-   Pouliot et al. Transplantation (2002) 73, 1751-7.-   Tajima and Pinnell. Biochem Biophys Res Commun. (1982) 106, 632-7.-   Weind et al. J Thorac Cardiovasc Surg (2002) 123, 333-40.-   Ye et al. European Journal of Cardio-thoracic Surgery (2000) 17,    449-454.

1. A method for preparing a human or animal tissue from at least onesheet of living tissue, the method comprising the steps of: (a)arranging said at least one sheet of living tissue to form a multi-layerstack of living tissue; and (b) applying a compressive force in adirection normal to the surface of the multi-layer stack of livingtissue with a force-applying means at a pressure and for an amount oftime sufficient to compress layers of tissue together for inducingadjacent layers of tissue to fuse or adhere to each other, wherein saidforce-applying means in step (b) comprises an adjustable weighted devicesuitable for applying substantially evenly-distributed pressure to saidmulti-layer stack of living tissue, said weighted device being at leastpartially permeable to tissue-culture medium.
 2. The method of claim 1,wherein said multi-layer stack is arranged on a substantially flatsupport.
 3. The method of claim 1, wherein said multi-layer stack ofliving tissue in step (a) is formed by superimposing two or more sheetsof living tissue.
 4. The method of claim 1, wherein said multi-layerstack of living tissue is formed by folding a sheet of living tissueupon itself.
 5. The method of claim 1, further comprising a step ofanchoring said multi-layer stack of living tissue with anchoring meansbefore said step (b) of applying a force, wherein said anchoring meansapplies sufficient tension across said multi-layer stack of livingtissue to prevent shrinkage and/or maintain cellular differentiationand/or induce fiber orientation.
 6. The method of claim 5, wherein saidanchoring means comprises a multiplicity of spaced apart weights oringots arranged substantially around the perimeter of said multi-layerstack of living tissue.
 7. The method of claim 1, wherein themulti-layered stack of living tissue in step (a) is formed by rolling asheet of living tissue on a tubular support.
 8. The method of claim 7,wherein said force-applying means in step (b) comprises a tissue-culturemedium permeable elastic sleeve.
 9. The method of claim 1, wherein cellsare obtained from said at least one sheet of living tissue, said atleast one sheet of living tissue is obtained via biopsy.
 10. The methodof claim 1, wherein said at least one sheet of living tissue is obtainedby culturing cells in vitro.
 11. The method of claim 10, wherein saidcells are selected from the group consisting of embryonic stem cells,post-natal stem cells, adult stem cells, mesenchymal cells, hepatocytes,islet cells, parenchymal cells, osteoblasts and other cells forming boneor cartilage, and nerve cells.
 12. The method of claim 11, wherein saidmesenchymal cells are selected from the group consisting of fibroblasts,interstitial cells, endothelial cells, smooth muscle cells, skeletalmuscle cells, myocytes, chrondocytes, adipocytes, fibromyoblasts, andectodermal cells.
 13. The method of claim 1, wherein said at least onesheet of living tissue is selected from the group consisting of a skintissue, a corneal tissue, a cardiac valve tissue, a connective tissueand a mesenchymal tissue.
 14. A multi-layer tissue made according to themethod of claim 1, wherein said multi-layer tissue comprises at leasttwo different types of sheets of living tissue.
 15. A multi-layer tissueaccording to claim 14, consisting essentially of between two sheets andtwelve sheets of living tissue.
 16. A multi-layer tissue according toclaim 14, consisting essentially of between three sheets and nine sheetsof living tissue.
 17. A multi-layer tissue according to claim 14,wherein said tissue has a thickness of about 0.01 mm to about 0.5 mm.18. A multi-layer tissue according to claim 17, wherein said tissue hasa thickness of about 0.03 mm to about 0.45 mm.